薬剤性内耳障害におけるプログラム細胞死の役割

学位の種別: 論文博士審査委員会委員 : (主査)東京大学教授 田之倉 優, 東京大学准教授 戸塚 護, 東京大学准教授 八村 敏志, フロリダ大学助教授 染谷 慎一, フロリダ大学准教授 永田 宏次University of Tokyo(東京大学

A potentiometric biosensing system based on an isolated degrading bacterium Klebsiella sp MP-6 for the determination of methyl parathion

A potentiometric sensing system for the sensitive and selective detection of methyl parathion (MP) is described in this paper. The system is based on a degrading bacterium Klebsiella sp. MP-6 as recognition element and a polymeric membrane anion-sensitive electrode as a transducer. Klebsiella sp. MP-6 can be isolated from long-term organophosphorus pesticide contaminated soils, which is capable of biodegrading MP to produce p-nitrophenol. The product can be deprotonated under basic conditions and thus detected by using the anion exchanger based membrane electrode. The bioreactor is prepared by packing the bacterial cells between two polyether sulfone membranes placed in a holder. Molecularly imprinted solid-phase extraction using the MP imprinted polymer as a sorbent enables accumulation and separation of MP from real samples. Under the optimized experimental conditions, the potential response of the biosensing system is linear with the MP concentration in the range of 5-100 nM. The detection limit is 1 nM. The electrode exhibits an excellent selectivity towards other organophosphorus pesticides. The sensing system has been evaluated with spiked water samples and shows good recovery and high accuracy. This methodology is promising to develop potentiometric sensors for detecting organophosphorus pesticides at trace levels in the environment.A potentiometric sensing system for the sensitive and selective detection of methyl parathion (MP) is described in this paper. The system is based on a degrading bacterium Klebsiella sp. MP-6 as recognition element and a polymeric membrane anion-sensitive electrode as a transducer. Klebsiella sp. MP-6 can be isolated from long-term organophosphorus pesticide contaminated soils, which is capable of biodegrading MP to produce p-nitrophenol. The product can be deprotonated under basic conditions and thus detected by using the anion exchanger based membrane electrode. The bioreactor is prepared by packing the bacterial cells between two polyether sulfone membranes placed in a holder. Molecularly imprinted solid-phase extraction using the MP imprinted polymer as a sorbent enables accumulation and separation of MP from real samples. Under the optimized experimental conditions, the potential response of the biosensing system is linear with the MP concentration in the range of 5-100 nM. The detection limit is 1 nM. The electrode exhibits an excellent selectivity towards other organophosphorus pesticides. The sensing system has been evaluated with spiked water samples and shows good recovery and high accuracy. This methodology is promising to develop potentiometric sensors for detecting organophosphorus pesticides at trace levels in the environment

Market, Policy, and Environmental Impact Analyses of a Novel 3D-Printed Concrete Marine Pumped Hydroelectric Storage Technology

While global extrication from humanity’s fossil fuel dependency is critical, the United States' clean energy transition cannot take place without a long duration energy storage solution operating in tandem with renewable energy development. Particularly with the generation of offshore wind energy, there are no commercially available energy storage technologies suited for turbine integration, grid connection, or the marine environment. Thus, our research investigated and evaluated the disruptive marine-pumped hydroelectric storage (M-PHES) technology of RCAM Technologies: a start-up based in Los Angeles, California that uses proprietary 3D concrete printing to create novel energy storage spheres for pairing with offshore wind. Our project scope included developing a revenue model for the commercial application, identifying ideal markets for pilot installations, outlining the regulatory implications and permitting processes involved in going to market, creating relevant geospatial visualizations, and assessing the potential environmental impact of the device on marine life as well as the impact on various stakeholders. In addition, our team attended the Business Network for Offshore Wind’s International Partnering Forum in 2022 to meet with industry experts and serve as an informal networking extension of the RCAM team, where they were being recognized as an innovative start-up. Our recommendations were utilized in multiple real-time funding proposals and grants for which RCAM was competing, and will continue to inform the company's strategy going forward.Master of Science (MS)School for Environment and SustainabilityUniversity of Michiganhttp://deepblue.lib.umich.edu/bitstream/2027.42/176193/1/Impact Assessment of RCAM’s M-PHES Technology on Marine Life, Water Quality, and Geology.pd

The miR-183/Taok1 target pair is implicated in cochlear responses to acoustic trauma.

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process

Effects of Cdh23 single nucleotide substitutions on age-related hearing loss in C57BL/6 and 129S1/Sv mice and comparisons with congenic strains.

A single nucleotide variant (SNV) of the cadherin 23 gene (Cdh23(c.753A)), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23(c.753A\u3eG) and 129S1-Cdh23(c.753G\u3eA)). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23(Ahl+) and 129S1.B6-Cdh23(ahl)) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23(c.753G) allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23(c.753A) allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23(c.753A) SNV and 129S1.B6-Cdh23(ahl) congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23(c.753)alleles. Importantly, the B6-Cdh23(c.753G)strain can be used to eliminate the confounding influence of the Cdh23(c.753A)variant in hearing studies of B6 mice and mutant mice on the B6 background. Sci Rep 2017 Mar 13; 7:44450

Nitric oxide synthase inhibitor reduces airway inflammation in mouse models with asthma

Objective To explore the therapeutic effect of nitric oxide synthase (NOS) inhibitor in asthmatic mice. Methods BALB/c mice of 5 to 6 weeks were randomly divided into five groups with 10 in each: 1)control group; 2)asthma group: The asthma model was established by ovalbumin (OVA)-sensitized mice; 3)S-methyl isothiourea sulfate (SMT) group: OVA-sensitized asthmatic mice were stimulated and then given with SMT; 4)N-nitro-L-arginine methyl ester (L-NAME) group: OVA-sensitized asthmatic mice were stimulated and then given with L-NAME; 5) NG-monomethyl-L-arginine (L-NMMA) group: OVA-sensitized asthmatic mice were stimulated and then given with L-NMMA. HE staining was used for histopathological analysis. Airway reactivity was assessed using a methacholine challenge test. The levels of nitric oxide (NO) and cytokines in serum and bronchoalveolar lavage fluid(BALF) were detected by ELISA. Results Compared with the control group, goblet cell proliferation and increased mucus secretion were found in the asthma group, together with a large number of inflammatory cell infiltration around the trachea and blood vessels and significantly increased airway resistance, which were improved after the administration of NOS inhibitors, especially in the SMT group. Compared with the control group, the levels of NO in plasma, BALF and lung tissue homogenate were significantly increased in the asthma group (P＜0.05), while the level of IL-2, IL-4, IL-17 and TNF-α in plasma and BALF was significantly increased (P＜0.05). Compared with the asthma group, the level of NO in plasma, BALF and lung tissue homogenate was significantly decreased in SMT group (P＜0.05), while the level of IL-2, IL-4, IL-17 and TNF-α in plasma and BALF was significantly decreased in SMT group (P＜0.05). Compared with the asthma group, the level of plasma NO, IL-2, IL-17, and TNF-α in the L-NAME group was significantly decreased (P＜0.05). The level of NO, IL-2, IL-17, and TNF-α in BALF was significantly decreased (P＜0.05). Compared with the asthma group, the plasma level of NO, IL-2, IL-4, IL-17 and TNF-α in the L-NMMA group was significantly decreased (P＜0.05). The level of NO, IL-2, IL-4, IL-17 and TNF-α in BALF was significantly decreased(P＜0.05), and the level of NO in lung tissue homogenate was significantly decreased (P＜0.05). Conclusions NOS inhibitor, especially SMT may reduce airway inflammation of asthmatic mice

Presbiacusia: dal laboratorio alla clinica. Attuali evidenze sperimentali e prospettive future

La perdita di udito legata all'età, o presbiacusia, colpisce circa il 35% dei soggetti sopra i 70 anni e rappresenta il secondo disturbo più comune tra gli anziani. La gravità della perdita uditiva può essere maggiore quando la valutazione audiometrica viene effettuate in condizioni più realistiche, come ad esempio in presenza di un rumore di fondo. I dati emergenti da alcuni studi nell’uomo e, soprattutto, nel modello animale suggeriscono che i danni alle cellule ciliate interne e/o ai neuroni di tipo I potrebbero essere responsabili di questo peggioramento del deficit uditivo in presenza di rumore di fondo. Pertanto, l’udito misurato con l’aggiunta di rumore potrebbe rappresentare un metodo efficace per individuare alterazioni in specifiche regioni frequenziali. È stato dimostrato nel modello animale che la somministrazione di N-acetilcisteina svolge un ruolo protettivo rilevante nei confronti delle cellule ciliate interne e dei neuroni di tipo I nei comuni processi dell’invecchiamento; tale approccio potrebbe avere un ruolo anche nell’uomo.Age-related hearing loss, which affects roughly 35% of those over the age of 70, is the second most common disorder among the elderly. The severity of age related hearing loss may actually be much worse when assessments are made under more realistic conditions such as communicating in noise. Emerging data from humans and animal models suggest that damage to the inner hair cells and/or type I neurons that relay sound information to the brain may contribute to hearing deficits in a noisy background. Tone detection thresholds measured in broadband noise may provide an efficient method of detecting the deficits in specific frequency regions. N-acetyl cysteine supplementation which protects against inner hair cell loss in animal models, may represent a viable therapy for protecting the inner hair cells and type I neurons

Cisplatin-induced vestibular hair cell lesion-less damage at high doses

Cisplatin, a widely used anticancer drug, damages hair cells in cochlear organotypic cultures at low doses, but paradoxically causes little damage at high doses resulting in a U-shaped dose-response function. To determine if the cisplatin dose-response function for vestibular hair cells follows a similar pattern, we treated vestibular organotypic cultures with doses of cisplatin ranging from 10 to 1000 μM. Vestibular hair cell lesions progressively increased as the dose of cisplatin increased with maximum damage occurring around 50–100 μM, but the lesions progressively decreased at higher doses resulting in little hair cell loss at 1000 μM. The U-shaped dose-response function for cisplatin-treated vestibular hair cells in culture appears to be regulated by copper transporters, Ctr1, ATP7A and ATP7B, that dose-dependently regulate the uptake, sequestration and extrusion of cisplatin. Keywords: Cisplatin, Ototoxicity, Copper transporters, Vestibular organotypic culture

Hidden age-related hearing loss and hearing disorders: current knowledge and future directions

Age-related hearing loss, which affects roughly 35% of those over the age of 70, is the second most common disorder among the elderly. The severity of age-related hearing loss may actually be worse if assessments are made under more realistic conditions, such as communicating in noise. Emerging data from humans and animal models suggest that damage to the inner hair cells and/ or type I neurons that relay sound information to the brain may contribute to hearing deficits in a noisy background. Data obtained from carboplatin-treated chinchillas suggest that tone-in-noise thresholds are a sensitive and frequency dependent method of detecting damage to the IHC/type I system. Therefore, tone detection thresholds measured in broadband noise may provide an efficient method of detecting the deficits in specific frequency regions. Preliminary data obtained in elderly subject with normal thresholds in quiet compared to young subjects illustrate the importance of repeating these measurements in broadband noise because thresholds in noise were worse for our elderly subjects than young subjects, even though both groups had similar hearing thresholds in quiet. N-acetyl cysteine supplementation which protects against inner hair cell loss in animal models may represent a viable therapy for protecting the inner hair cell/type I neurons